Project Summary Approximately 90% of individuals infected with Mycobacterium tuberculosis (Mtb) develop an asymptomatic latent infection, which is non-infectious. Although some individuals may eradicate this infection, those who do not comprise a large reservoir of persons who can convert from latent to active TB, which is infectious. Reactivation is especially likely in immune-compromised individuals, including those infected with HIV. The molecular switches that enable Mtb to slow or stop replication, become dormant and establish latent TB infection are poorly characterized. A thorough understanding of these switches is critical for development of 1) diagnostics to enable prediction of reactivation risk and 2) shorter, more effective treatment regimens for latent TB infection. Toxin-antitoxin (TA) systems are strongly implicated in establishment of latent TB infection because their toxin components typically downregulate Mtb cell growth and are activated in response to stresses relevant to this state. Yet, the extraordinary redundancy of TA systems make determination of the individual contributions of each toxin challenging using conventional genetic and molecular biological approaches. We propose to use a powerful battery of genome-scale tools to track the fate of transcripts, ribosomes and proteins in response to activation of a subset of tRNA-cleaving toxins to understand the molecular mechanisms that underlie stress survival. We then exploit our finding that the codon-specific ribosome-stalling characteristic of these toxins identifies novel ORFs and apply this as a reliable tool for improved Mtb genome annotation.